The overall aim of this project is to determine by X-ray crystallography the precise molecular architecture of certain alpha-proteins that have dynamic as well as structural roles. Muscle proteins are a central focus and provide the background for studying related systems. A major aim is to characterize the strong actin-bound rigor conformation of myosin, as well to understand how myosin is regulated in scallop striated and (the closely related) vertebrate smooth muscles. All conventional myosin head (S1) crystal structures are thus far found in weak actin-binding states. Actin trimers and mini-thin filaments (MTF) are being prepared for crystallization in complex with S1 so that the strong actin binding state of the contractile cycle may be visualized. The trimers and MTFs will also be crystallized alone to provide a high resolution structure of undecorated F-actin. Structural investigations of myosin will also include studies of S1 with inhibitors of its ATPase activity, as well as various regions of the long myosin tail. Double headed constructs of myosin will be produced and studied in the "off" state in order to understand myosin-linked regulation of contraction. Our second aim is to visualize atomic structures of the tropomyosin/troponin switch, which controls contraction in vertebrate striated muscles. These investigations will extend the recently completed structure determinations of the N- and C-termini of tropomyosin to the molecule's middle segments;also, novel peptide constructs will be used to obtain the structure of the critical head-tail overlap region that allows the filament to form. The third aim, in a related area, is the analysis of fibrinogen/fibrin assembly. New central fragments of the molecule will be used in attempts to crystallize the DDE (-like) ternary nucleus of fibrin. Another goal is the structure determination of fibrinogen variants that underlie certain clotting disorders. A final aim is the analysis of design motifs in the alpha-helical coiled coil, especially in large fibrous proteins whose structures we are determining. We are convinced that detailed knowledge of the molecular mechanisms of muscle contraction and of the Ca2+-controlled tropomyosin/troponin switch is essential in order to correct malfunctions in various muscle diseases. Without atomic structures of these muscle proteins in different physiological states, the significance of disease producing mutations cannot be understood, nor can there be rational intervention at the molecular level directed to overcoming functional defects in these molecules. Similarly, in order to understand and control both normal and impaired clot formation, it is essential to have detailed structural information about the molecules and their interactions in the clot. A deeper understanding of the factors controlling the conformation, stability and partner selection in alpha-helical coiled coils will allow the design of therapeutic and diagnostic peptides targeted to naturally occurring coiled coil motifs.